Tissue plasminogen activator (tPA) is an extremely promising drug which can be used to dissolve blood clots, most notably in heart attack victims. It is a fibrin-dependent serine protease which catalyzes the conversion of plasminogen to plasmin in one of the essential steps in the fibrinolytic cascade. The tPA enzyme was initially prepared from a variety of mammalian cell types, and was initially purified from human uterus (Rijken, D.C. et al. Biochem Biophys Acta (1979) 580:140-153) and from the Bowes melanoma cell line (Rijken, D.C. et al. J Biol Chem (1981) 256:7035-7041).
tPA is also known to be secreted naturally from a number of tissue sources including pig heart, fetal kidney, lung, and colon fibroblast cells. Recently, tPA has been produced using recombinant means by a number of groups, initiated by the successful cloning of the cDNA by Pennica, D. et al. Nature (1983) 301:214-221. The protein can be produced in a variety of hosts including E. coli, mouse L cells, CHO cells and yeast. (See, for example, EP Publication No. 174,835 (UpJohn), EP Publication No. 161,935 (Eli Lilly), EP Publication No. 143,081 (Ciba-Geigy), PCT application No. W086/05514 (Chiron).) Native tPA has been produced as disclosed by Snow Brand Milk Products (EP 196,226); Kochi Medical School (EP 194,736); Kowa KK and Asahi (EP 151,996); Meiji Milk Products (GB 2,153,366); Choay, S.A. (EP 133,070) and Asahi and Kowa KK (U.S. 4,505,893); and by Wakamoto Pharmaceutical (Biotechnology, Nov. 1986). Also relevant are two Genentech applications describing the production of tPA in CHO cells (EP 117,059 and EP 117,060).
Regardless of the manner in which tPA is produced, it is necessary to solubilize the protein to a workable concentration in aqueous solutions in order to administer small enough volumes to allow prompt administration of an effective dose. An effective dose is expected to be in the range of 1.times.10.sup.6 -5.times.10.sup.7 IU; highly purified tPA is expected to have a specific activity on the order of 2-5.times.10.sup.5 IU per mg. Thus, in order to administer as little as 1 ml of a pharmaceutical composition, the solubility must be increased to at least about 5 mg per ml, and preferably to 50 mg per ml.
In addition, efficient purification of tPA produced by any of the foregoing cells requires a series of chromatographic procedures which are more efficiently conducted if a high level of solubility is maintained. For adaptation of purification procedures to tPA-containing medium, it would be desirable to achieve solubilities in these same ranges.
A variety of formulations for tPA has already been proposed. Many such formulations are concerned with stabilizing the tPA rather than enhancing its solubility. The proposed formulation most closely related to that disclosed hereinbelow is that set forth in EPO application Publication No. 217,379, published 8 Apr. 1987 to Mochida Pharmaceutical Co. Ltd. The formulations proposed are in arginine solution or a solution containing arginine and an acid addition salt. It appears that solubilities of the order of 10 mg/ml are achieved at arginine hydrochloride concentrations of approximately 0.5 M and above. As the arginine hydrochloride concentration is lowered, the solubility is diminished so that at 0.025 M arginine hydrochloride, only about 2 mg/ml dissolves. In the absence of arginine, in saline solution, less than 0.1 mg/ ml solubility is exhibited. The pH of the formulation is not specified, but it is assumed to be that generated by arginine hydrochloride at the specified concentration.
The Mochida application also suggests that solubilities of the order of 10 mg per ml can be achieved at pH 7 in the presence of 0.1 M arginine hydrochloride and that somewhat higher concentrations can be achieved by supplementing the solution with high concentrations of salt. At lower pH (pH 2, pH 4) and higher pH (pH 9, pH 11), at lower arginine concentrations (0.25 M) comparable or diminished solubilities were found. The pH 4 solution was obtained by adjusting the arginine solution with citric acid.
Based on the Mochida disclosure, it would appear that the upper solubility limit in 0.1 M arginine is approximately 5 mg/ml.